Proximal, selective, and dynamic interactions between integrin αIIβ3 and protein tyrosine kinases in living cells

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in alphallbbeta3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these alphallbbeta3-tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that alphallbbeta3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of alphallbbeta3-c-Src, alphallbbeta3-Syk, and c-Src-Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with alphallbbeta3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin beta3 cytoplasmic tail. Thus, alphallbbeta3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of alphallbbeta3 signaling to the actin cytoskeleton.